Splice variants of brca1 and brca2

ABSTRACT

Nucleic acid sequences encoding splice variants of BRCA1 and BRCA2 and uses thereof are provided.

INTRODUCTION

[0001] This invention was made in the course of research sponsored by the National Institutes of Health and the U.S. Army medical research. The U.S. Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] BRCA1 and BRCA2 are tumor suppression genes shown to be involved in 90% of familial breast and ovarian cancers (Miki et al. Science (1994) 266, 66-71). The full length gene sequence of BRCA1 is disclosed by Miki et al. The full length gene sequence of BRCA2 is deposited in GenBank, Accession Number U43746. Mutations in the breast and ovarian cancer susceptibility gene, BRCA7, account for about half of the inherited breast and ovarian cancers (Miki et al. (1994) Science, 266, 66-71; Easton et al. (1995) Am. J. Hum. Genet., 56, 265-271; Ford et al. (1995) Am. J. Hum. Genet., 57, 1457-1462) and about 10% of the sporadic ovarian cancers (Futreal et al. (1994) Science, 266, 12-122; Hosking et al. (1995) Nature Genet., 9, 343-344; Merajver et al. (1995) Nature Genet., 9, 439-443). BRCA2 was found to be associated more frequently with male breast cancer compared to BRCA1 (Wooster, R. et al. 1994. Nature 265:2088-2090. Patients with BRCA2 mutations were also found to be at higher risk for a variety of other cancers including carcinomas of the pancreas, prostate, and colon (Thorlacius, S. et al. 1996. Nat. Genet. 13:117-119; Phelan, C. M. et al. 1996. Nat. Genet. 13:120-122; Gudmundsson, J. et al. 1995. Cancer Res. 55:4830-4832; Tonin, P. et al. 1995. J. Med. Genet. 32:982-984).

[0003] The BRCA1 cDNA codes for a 1863 amino acid protein with an amino terminal zinc ring finger domain and a carboxy terminal acidic region (Miki et al. 1994 Science, 266, 66-71) typical of several transcriptional factors. The BRCA2 gene is composed of 27 exons and encodes a protein of 3418 amino acids with no significant homology to any known protein (Wooster, R. et al. 1995. Nature 378:789-792; Bork, P. et al. 1996. Nat. Genet. 13:22-23). Recently, the C terminal region of BRCA1 was shown to activate transcription in a heterologous GAL-4 system (Chapman, M. S. and Verma, I. M. (1996) Nature, 382, 678-679; Monteiro et al. (1996) Proc. Natl. Acad. Sci. USA, 93, 13595-13599). Murine BRCA1 has been cloned and the developmental patterns of expression studied (Lane et al. (1995) Genes and Development, 9, 2712-2722; Marquis et al. (1995) Nature Genetics, 11 17-26; Abel et al. (1995) Hum. Molec. Genet., 4, 2265-2273; Sharan et al. (1995) Hum. Mol. Gen., 4, 2275-2278). Expression was found to be high in rapidly proliferating tissues (Lane et al. (1995) Genes and Development, 9, 2712-2722; Marquis et al. (1995) Nature Genetics, 11 17-26), particularly those undergoing differentiation, thus suggesting a role for BRCA1 in cellular growth and differentiation. The BRCA1 gene product has been shown to be a nuclear phosphoprotein (Chen et al. (1995) Science, 270, 789-791; Rao et al. (1996) Oncogene, 12, 523-528; Scully et al. (1996) Science, 272, 123-125) that, when overexpressed in breast and ovarian cancer cells, results in growth inhibition in vitro and in vivo. Conversely, inhibition of BRCA1 expression by antisense RNA in mouse fibroblasts or by antisense oligonucleotides in breast cancer cells resulted in transformation of mouse fibroblasts as well as an increase in the rate of growth of breast cancer cells (Thompson et al. (1995) Nature Genetics, 9, 444-450; Rao et al. (1996) Oncogene, 12, 523-528).

[0004] Two new alternately spliced BRCA1 transcripts referred to as BRCA1a and BRCA1b have recently been isolated and mouse fibroblast cell lines and human breast cancer cell lines expressing BRCA1a proteins have been developed (Shao et al. (1996) Oncogene, 13, 1-7). Overexpression of BRCA1a was found to induce apoptosis in NIH3T3 and MCF-7 cells after calcium ionophore treatment thus indicating that BRCA1 proteins, specifically BRCA1a, play a role in the regulation of apoptosis (Shao et al. (1996) Oncogene, 13, 1-7). The role of BRCA2 in apoptosis remains to be elucidated.

[0005] Two proteins, BARD1 and Rad51 which are human homologs of bacterial Rec A, were shown to interact both in vitro and in vivo with BRCA1 and BRCA2 indicating a role for BRCA1 proteins in tumor suppression and a role for BRCA1 in the control of recombination and genomic integrity, as well as a role for BRCA2 in DNA repair (Wu et al. (1996) Nat. Genetics, 14, 430-447; Scully et al. (1997) Cell, 88, 265-275; Sharan, S. K. et al. 1997. Nature 386:804-810; Zhang, H- T. et al. 1998. Cell. 92:433-436).

[0006] Previously, the BRCA1 gene product was shown to be localized in the nucleus (Chen et al. (1995) Science, 270, 789-791; Rao et al. (1996) Oncogene, 12, 523-528). However, differences regarding the size and subcellular localization of BRCA1 have been reported (Chen et al. (1995) Science, 270, 789-791; Chen et al. (1996) Science, 272, 125-126; Jensen et al. (1996) Nature Genetics, 12, 303-308; Scully et al. (1996) Science, 272, 123-125; Thakur et al. (1997) Mol. Cell. Biol., 17, 444-452; Wilson et al. (1997) Oncogene, 14, 1-16). Two additional BRCA1 splice variants, BRCA1Δ672-4092 (which lacks exon 11) and BRCA1Δ11b (which lacks a majority of exon 11) were recently found to localize to the cytoplasm by immunostaining (Thakur et al. (1997) Mol. Cell. Biol., 17, 444-452; Wilson et al. (1997) Oncogene, 14, 1-16). BRCA1Δ11b was also found to be present in significant quantities in the nuclear fractions on immunoblotting analysis.

[0007] Both BRCA1 and BRCA2 gene products have been reported to be regulated in a cell cycle-dependent manner and to have a potential transactivation function (Rajan, J. V. et al. 1996. Proc. natl. Acad. Sci. USA 93:13078-13083; Vaughn et al. (1996) Cancer Res. 56, 4590-4594; Chapman, M. S. and I. M. Verma. 1996. Nature 382:678-679; Monteiro, N. A. et al. 1996. Proc. Natl. Acad. Sci. USA 93:13595-13599; Milner, J. et al. 1997. Nature 386:772-773; Cui, J. et al. 1998 Oncology Reports 5:585-589).

SUMMARY OF THE INVENTION

[0008] The sequences of BRCA1a and BRCA1b, two splice variants of BRCA1, have now been determined. It has been found that BRCA1a lacks a majority of exon 11 (amino acids 263-1365) while and BRCA1b lacks exons 9, 10 and a majority of exon 11 (amino acids 263-1365). Like BRCA1, BRCA1a encodes a phosphoprotein containing phosphotyrosine that associates via its amino-terminal zinc ring finger domain with E2F transcriptional factors, cyclin and cyclin dependent kinase (cdk) complexes. The amino-terminal region of BRCA1a has now been demonstrated to function as a transactivation domain when fused to heterologous GAL4 DNA binding domain. Additional studies indicate the presence of a negative regulatory domain at the carboxy-terminal regions of BRCA1 and BRCA1a proteins. It is believed that mutations in the zinc ring domain found in patients with breast and ovarian cancer may impair this activity, thus indicating that a loss of transcriptional activation by BRCA1 may lead to the development of breast and ovarian cancers.

[0009] The protein encoded by the BRCA1b splice variant, however, has now been found to have lost a portion of the amino-terminal transactivation domain as a result of alternate splicing. Accordingly, it is believed that BRCA1b may function as a dominant-negative regulator of the transcriptional activation function of BRCA1/BRCA1a proteins.

[0010] BRCA1 encoded proteins have now been found to accumulate in the cytoplasm in the presence of serum and in the nucleus in the absence of serum thus indicating that the nuclear localization of BRCA1 may be regulated by external stimuli, phosphorylation or protein-protein interactions. Like BRCA1, proteins encoded by BRCA1a and BRCA1b both act as tumor suppressors, thus indicating that exon 11 is not need for this function. Proteins which interact with BRCA1 have also been identified.

[0011] It has also been found that the amino terminal region of BRCA2, like BRCA1, associates with transcriptional factor E2F, cyclin and cdk's. BRCA2 has also been found to undergo differential splicing. The sequence of a BRCA2 splice variant, BRCA2a, has now been determined and contains a deletion of a putative transcriptional activation domain, giving rise to a protein with potential dominant negative pathophysiology. In addition, experiments examining the transcriptional factor function of BRCA2 demonstrate that the BRCA2 proteins have intrinsic histone acetyl transferase activity, activity that maps to the amino-terminal region of BRCA2. The BRCA2 proteins acetylate mainly H3 and H4 of free histones.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 shows the structure of the splice variants BRCA1a and BRCA1b. “BNT” is used to designate the transactivation domain at the amino-terminal region, while “BID” is used to designate the inhibitory domain at the carboxy-terminal region of the protein.

[0013]FIG. 2 shows the structure of the splice variant BRCA2a .

DETAILED DESCRIPTION OF THE INVENTION

[0014] BRCA1 gene products are nuclear tyrosine phosphoproteins that have now been found to translocate to the nucleus in the absence of serum. These phosphoproteins associate in vitro with E2F transcriptional factors, cyclins and cdk complexes. The structure and nucleotide sequences of two splice variants of BRCA1, BRCA1a and BRCA1b (FIG. 1) have now been determined and the activity of proteins encoded thereby analyzed.

[0015] Full length cDNAs and several amino and carboxy-terminal deletions of BRCA1a and BRCA1b were subcloned in-frame with the GAL4 DNA binding domain vector (Webster et al. Cell (1988) 52, 169-178). The GAL4 fusion constructs were co-transfected in NIH3T3 fibroblast cells, along with the reporter plasmid 17MX2-tk-CAT that contains two GAL4 binding sites linked to the chloramphenicol acetyltransferase gene (CAT) and an internal control plasmid CH1210. Both full length GAL4-BRCA1a and GAL4-BRCA1b plasmids failed to show CAT activity when compared to the GAL4 vector control. Two carboxy-terminal truncated BRCA1a plasmids, one lacking exons 21-24 and the other lacking exons 16-24, failed to show transactivation. Further carboxy-terminal deletion of BRCA1a (deletion of amino acids 263-1863) but not BRCA1b plasmid (deletion of amino acids 263-1863) showed approximately 7-10 fold CAT activity. Further carboxy-terminal deletions of exons 9 and 10 of BRCA1a and exons 9 and 10 alone when fused to the GAL4 DNA binding domain failed to show significant activity thus indicating that additional sequences N-terminal and C-terminal to this region are required for transcriptional activation.

[0016] Thus, like BRCA1, the protein encoded by the BRCA1a splice variant has a transactivation domain which include the intact zinc ring finger domain at the amino-terminal regions. This domain has been shown to interact both in vitro and in vivo with BARD1 and in vitro with E2Fs, cyclins and cdk complexes and thus is important in mediating protein-protein interactions. A negative regulatory domain at the carboxy-terminal regions of both BRCA1 and BRCA1a proteins was also identified.

[0017] The BRCA1 zinc ring finger domain is the location of some of the most frequently occurring mutations linked to breast and ovarian cancers. For example, one of the BRCA1 mutations contains a frame shift in exon 2 (188 del 111; Miki et al. (1994) Science, 266, 66-71) which removes the zinc ring finger domain from the protein. The 185 del AG mutation, which is the most common BRCA1 mutation seen to date that occurs in 1 in 100 Ashkenazi Jewish individuals disrupts the BRCA1 gene product at the first residue of the C₃HC₄ domain (Shattuck-Eiden et al. (1995) JAMA, 15, 535-541). These frequent missense mutations Cys 61, Gly, Cys 64, Gly, and Cys 64 Tyr also disrupt the zinc ring finger domain structure. The lack of, or impaired binding of, the disrupted BRCA1 protein to E2F, cyclins and/or cdk's in patients with mutations in the zinc ring finger domain is believed to result in a loss of transcriptional activation by BRCA1, thus depriving the cell of an important mechanism for regulating cell proliferation and leading to the development of breast or ovarian cancer.

[0018] The protein encoded by the BRCA1b splice variant has also lost a portion of the amino-terminal transactivation domain as a result of alternate splicing. It is believed that the BRCA1b protein may function as a dominant-negative regulator of the transcriptional activation function of BRCA1/BRCA1a proteins. The subcellular localization of BRCA1a and BRCA1b proteins has now been determined using FLAG epitope tagged CMV promoter vectors. Data using immunofluorescence and immunoblotting analysis indicate cytoplasmic and nuclear localization of FLAG BRCA1a and FLAG BRCA1b proteins. In these experiments, the normal function of the BRCA1 protein was examined with polyclonal antibodies generated against different regions of the human BRCA1 protein. The subcellular distribution of BRCA1 in normal human mammary epithelial cell Hs578 Bst and several human breast cancer cell lines HBL-100, CAL-51, MDA MB-453, T-47D, BT-474, CAMA-1 and ZR 75-1 was determined by immunohistochemistry and immunofluorescence staining. BRCA1 was detected mainly in the cytoplasm with weak nuclear staining of normal breast epithelial cell Hs 578 Bst and several breast tumor cell lines HBL-100, MDA MB-453, T-47D, BT474, CAMA-1 and ZR 75-1. One cell line, CAL-51, which was originally obtained from a patient with invasive adenocarcinoma with extensive intraductal involvement (Gioanni et al. (1990) Br. J. Cancer, 62, 8-13), contained three distinct populations of cells, some in which BRCA1 was localized in the cytoplasm, some in the perinucleus and some in the nucleus.

[0019] The subcellular distribution of BRCA1 in two ovarian carcinoma cell lines NIH:OVCAR-3 and SK-OV-3 was also determined. In NIH:OVCAR-3 cells BRCA1 was localized mainly to the cytoplasm and in SK-OV-3 BRCA1 was localized mainly in the nucleus. The subcellular distribution of BRCA1 in several normal and tumor cells was also determined. BRCA1 was found to be distributed mainly in the cytoplasm of NIH3T3 mouse fibroblast cells and Saos-2 cells, both in the cytoplasm and nucleus of BRCA1a transfected NIH3T3 cells, HeLa, Colo 320, A431 and PC12 cells and mainly in the nucleus with weak cytoplasmic staining of BALB/3T3 cells. All these results suggested variable, subcellular distribution of BRCA1 proteins. These results were obtained using different BRCA1 antibodies. To determine whether the differences in the subcellular localization of BRCA1 resulted from splice mutations, differentially spliced human BRCA1 cDNA were subcloned. BRCA1a was inserted into the pFLAG-CMV-2 expression vector (Eastman Kodak Company, Rochester, N.Y.) which contains a FLAG epitope-tag sequence at the N-terminal thus allowing detection of BRCA1a protein with the use of FLAG antibody. When transfected into COS cells the FLAG BRCA1a protein was found to be localized mainly in the cytoplasm with weak nuclear staining. Subcellular fractionation of the transfected COS cells into total and nuclear fractions followed by Western blot analysis using the same FLAG antibody revealed two polypeptides migrating at 105-110 kD mainly in the cytoplasm with a small fraction of the total BRCA1a protein in the nuclear fractions. These results suggest BRCA1a p110 to be a localized both in the cytoplasm and nucleus. The doublet protein bands observed are believed to represent hyper and hypophosphorylated forms of BRCA1a proteins, similar to the 220 kDa BRCA1 protein (Chen et al. (1996) Cancer Res. 56, 3168-3172). Western blot analysis of FLAG immunoprecipitates obtained from p-FLAG-CMV-2-BRCA1a transfected COS cells using phosphotyrosine antibodies revealed BRCA1a to be a phosphoprotein containing tyrosine which migrated with mobility similar to an endogenous BRCA1 tyrosine phosphorylated band seen in HL60 cells. These results indicate that BRCA1a is an ≈110 kD phosphoprotein which contains tyrosine. Similarly, BRCA1b was found to be a phosphoprotein phosphorylated on tyrosine migrating with a molecular weight of ≈100 Kd.

[0020] The subcellular distribution of FLAG-BRCA1a in breast cancer cell line CAL-51 was determined by immunofluorescence analysis following transfection of pFLAG-CMV-2 BRCA1a plasmid into these cells. BRCA1a protein was observed to be distributed both in the cytoplasm as well as in the nucleus, but the cytoplasmic staining appeared to be much stronger compared to the weak nuclear staining.

[0021] To determine whether the nuclear localization of BRCA1 is dependent on the proliferation state of the cell, the subcellular distribution of BRCA1 in asynchronous and serum deprived NIH3T3 cells was determined using immunofluorescence and immunohistochemical methods with BRCA1-specific antibodies. In asynchronous serum fed NIH3T3 cells, BRCA1 was found to be localized predominantly in the cytoplasm with weak nuclear staining. In contrast, in serum starved quiescent cells most of the BRCA1 was found to be localized in the nucleus with little cytoplasmic staining. On prolonged incubation in serum free media for 48-72 hours the staining was mostly nuclear with typical nuclear dot like pattern. When the cells were refed with 10% serum for 24 hours the BRCA1 protein was found to relocate back to the cytoplasm with weak nuclear staining similar to the situation seen in asynchronous serum fed NIH3T3 cells. The same results were obtained using four different antibodies specific to different regions of BRCA1 protein, thus ruling out the possibility that nuclear BRCA1 immunofluorescence might be an artifact due to starvation. The DNA content of these cells was determined simultaneously in the presence and absence of serum by FACS analysis to give a measure of the relative percentage of G1, S, G2/M cells in each sample. Based upon these experiments, it appears that nuclear transport of BRCA1 is not cell cycle dependent and occurs irrespective of the cell cycle state. Instead, BRCA1 protein accumulates in the nucleus in the absence of serum and in the cytoplasm in the presence of serum, in NIH3T3 cells. Additional experiments were performed wherein cells were arrested in the G1 phase of the cell cycle using drugs such as aphidicolin, mimosine and double thymidine block. Results from these experiments suggest that the redistribution of the BRCA1 proteins to the nucleus in drug-treated cells is not as dramatic as seen with serum starved cells.

[0022] Similar experiments were performed in asynchronous and growth arrested serum deprived normal human breast epithelial cells Hs578 Bst, breast cancer cells HBL-100, ZR-75-1, CAMA-1 and ovarian carcinoma cells NIHOVCAR-3, using BRCA1 specific polyclonal antibodies. These cell lines include only those in which BRCA1 was found to be localized to the cytoplasm in asynchronous conditions. In these experiments, BRCA1 was found to be localized mainly in the cytoplasm of serum fed asynchronous normal breast epithelial cells Hs578 Bst, breast tumor cells HBL-100, ZR-75-1, CAMA-1 and ovarian carcinoma cell line NIHOVCAR-3 and predominantly in the nucleus of growth arrested serum deprived Hs578 Bst, HBL-100, ZR-75-1 CAMA-1 and ovarian carcinoma cell line NIHOVCAR-3. Thus, nuclear or cytoplasmic transport of BRCA1 does not appear to be spontaneous but rather is controlled by the extracellular environment with serum growth factors inhibiting the nuclear transport of the BRCA1 protein.

[0023] Two cellular proteins (p65 BIP, p32 BIP) that specifically interact with BRCA1 were detected and isolated. A fusion protein that contains GST and the zinc ring finger domain of BRCA1 (residues 1-76) was expressed in bacteria using the Gex 2T expression vector system. In order to detect cellular proteins that interact with GST-BRCA1 fusion protein, whole cell lysates of human breast cancer cells ZR-75-1 or CA1-51 metabolically labeled with [³⁵S] methionine were incubated with either GST or the GST-BRCA1 fusion protein immobilized on glutathione-agarose beads (GSH-beads). The beads were washed, lysed in SDS sample buffer and subjected to SDS-PAGE. SDS-PAGE analysis of the bound complex revealed bands with relative molecular weight of ≈32 kD and ≈65 kD which bound exclusively to the GST-BRCA1 fusion protein and not to GST. These bands were consistently detected in ZR-75-1, CAL-51 and HL 60 cell lysates and were designated as p65 BIP and p32 BIP -respectively.

[0024] Using Western blot analysis, both p65 BIP and p32 BIP were found to bind strongly to BRCA1 even under stringent conditions without any mediating proteins. In these experiments, the BIP complexes prepared from CAL-51 cell extract separated on SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with ³²P-labeled GST-TK-BRCA1 fusion protein. Since GST-TK-BRCA1 contains a consensus phosphorylation site for protein kinase C at the amino terminal end, the purified protein can be ³²P-labeled by an in vitro protein kinase reaction. GST-BRCA1 bound to both p65 BIP and p32 BIP unlike GST protein.

[0025] The distribution of BRCA1-binding protein in various cell lines was determined in metabolically labeled promyelocytic cell line HL 60, breast cancer cell lines ZR-75-1 and CAL-51 cells by the GST-pull down assay. Both p65 BIP and p32 BIP were detected in all cell lysates examined, although at variable levels suggesting the ubiquitous expression of p65 BIP and p32 BIP.

[0026] BIP complexes obtained from CAL-51 cells were immunoblotted with a phosphotyrosine antibody. A band corresponding to p32 was detected indicating that p32 BIP contains phosphotyrosine, a characteristic specific to cyclin associated protein kinases.

[0027] BRCA1 was also found to similarly associate with E2F cyclins and cdk's. Cell lysates obtained from CAL-51 cells were incubated with GST-immobilized on GSH-beads and BRCA1 fusion protein conjugated GSH-beads. The beads were then washed and heated in SDS sample buffer. The BIP complexes were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membrane and probed with antibodies specific to cdc2, cdk2, cdk3, cdk4, cdk5, cdk6, cyclin A, cyclin B1, cyclin D1, cyclin E, E2F-1, E2F-2, E2F-3, E2F-4 and E2F-5. The BIP complexes were recognized by antibodies specific to cdc2, cdk-2, cdk-4, cyclin D1, cyclin A, cyclin B1 and E2F-4, but not to cdk3, cdk-5, cdk-6, cyclin E, E2F-1, E2F-2, E2F-3 and E2F-5 thus indicating that BRCA1 associates with cyclin A, D1 and B1, cdc2, cdk-2, cdk-4, and -E2F-4, but not with cdk3, cdk-5, cdk-6, cyclin E, E2F-1, E2F-2 and E2F-5. BRCA1 immunoprecipitates from CAL-51 cells were tested for kinase activity. These immunocomplexes show histone H1 kinase activity confirming the association of BRCA1 with cyclins/cdk complexes.

[0028] To examine the binding of cyclin A, cyclin B1, cyclin D1, E2F-1 and E2F-4 with BRCA1a and BRCA1b in vitro, cDNA sequences encoding BRCA1a and BRCA1b were inserted into pcDNA3 expression vectors. In vitro transcription and translation of BRCA1a and BRCA1b in the presence of [³⁵S] methionine generated radiolabeled BRCA1a and BRCA1b polypeptides of approximately 110 and 100 kD respectively. These radiolabeled proteins were passed through GST-cyclin A, GST-cyclin B1, GST-cyclin D1, GST- E2F-1, GST-E2F-4 and GST respectively. Both in vitro translated BRCA1a and BRCA1b specifically bind to GST-cyclin A, GST-cyclin B1, GST-cyclin D1, GST-E2F-1 and GST E2F-4 unlike GST alone. However, the BRCA1a splice variant bound at a reduced level compared to BRCA1b to all these different proteins. Further GST and GST E2F-l fusion proteins were subjected to far Western Blot analysis using ³²P-labeled amino-terminal BRCA1 (GST-BRCA1a amino acids 1-76, numbering from first ATG codon) fusion protein. The GST E2F-1 fusion protein band hybridized specifically to BRCA1. To further confirm the results, a fragment of BRCA1 encoding the amino-terminal 182 amino acids (amino acids 1-182) was in vitro translated and assayed for binding to GST-E2F-1 protein. The BRCA1 polypeptides bound specifically to GST-E2F-1. In a reciprocal assay, the full length human E2F-1 CDNA was in vitro translated and assayed for binding to GST-BRCA1 zinc ring finger domain fusion protein (amino acids 1-76). The E2F-1 polypeptide bound very weakly to GST BRCA1. These results suggest that the amino-terminal 76 amino acids of BRCA1 were sufficient to provide specific association with E2F-1.

[0029] Similarly, human cdc2, cdk2, cdk4 and cdk5 were in vitro translated. The proteins thus generated were assayed separately for binding to GST and GST-BRCA1(amino acids 1-76) fusion proteins. GST-BRCA1 specifically bound to cdc2 and cdk2 but not to cdk4 and cdk5 thus indicating that BRCA1 zinc ring finger domain can interact directly with cdc2 and cdk2.

[0030] Accordingly, as demonstrated herein, proteins encoded by the BRCA1a and BRCA1b splice variants have a number of similar characteristics and activities related to the tumor suppressor protein BRCA1. However, the shorter length of the nucleotide sequences of these splice variants renders them more suitable for establishing permanent cell lines for expression of these tumor suppression genes. Such cells lines will serve as useful model systems for studying BRCA1 gene therapy in breast and ovarian cancer cells. Since BRCA1a induces apoptosis of breast cancer cells, these cells lines will also be useful in studying whether the rate of apoptosis increases significantly and leaves the cancer cells more susceptible to treatment with radiation and chemotherapeutic agents such as Taxol, cisplatin, etc. These cell lines will also be useful for understanding the chain of events and identifying downstream targets through which the BRCA1 proteins turn on cellular apoptosis.

[0031] Further, the shorter length of these splice variants makes inclusion of these nucleotide sequences into viral vectors such as retroviruses for use as gene therapy agents more feasible. Viral vectors containing a splice variant of the BRCA1 tumor suppressor gene, BRCA1a can be administered to a patient suffering from breast or ovarian cancer to increase levels of protein encoded by the nucleotide sequences thereby suppressing the tumor, or inhibiting proliferation or inducing apoptosis of breast or ovarian cancer cells.

[0032] A splice variant of BRCA2, BRAC2a, has also been cloned and characterized (FIG. 2). The amino terminal region of BRCA2, like BRCA1, was shown to associate with transcriptional factor E2F, cyclins and cdk's. Thus, it is believed that proteins encoded by the tumor suppression genes BRCA1 and BRCA2 interact with E2Fs and regulate cell proliferation. The protein encoded by BRCA2a, however, has lost the transcriptional activation domain and is believed to compete with native BRCA2 in terms of DNA binding or interaction with other transcriptional factors resulting in a dominant negative effect on the transcription activation function of BRCA2.

[0033] The function of the BRCA2 tumor suppression gene was examined by cloning several cDNAs by RT-PCR and characterizing these CDNA by nucleotide sequence and restriction map analysis. It was found that one of the cDNAs (referred to herein as BRCA2a) showed alternate splicing resulting in the deletion of exon 3. Previously, this exon was shown to contain a potential transcriptional activation domain thus suggesting that BRCA2 may. function as a transcription activator (Milner et al. (1997) Nature 386, 772-773). Since BRCA2a has lost the transcriptional activation domain, it is believed to compete with native BRCA2 in terms of DNA binding or interaction with other transcriptional factors resulting in a dominant negative effect on the transcription activation function of BRCA2. Similar dominant negative variants have been observed in other transcriptional activators (Foulkes et al. (1991) Cell 64, 739-749).

[0034] Further, in GST-pulldown assays, in vitro translated [³⁵S]-methionine labelled BRCA2 (aa 1-500) was bound to GST-cyclin A, GST-cyclin B1, GST-cyclin DA and GST-E2F-1, but not GST itself. These results indicate that BRCA2, like BRCA1, associates with E2F-1, cyclin B1 and cyclin D1, thereby negatively regulating the cell cycle. The ability of the alternatively spliced variant, BRCA2a to bind to these regulatory factors was also tested. BRCA2a was found to also bind to GST-cyclin A, GST-cyclin B1, GST-cyclin D1 and GST-E2F-1, indicating that exon 3 is not needed for these interactions. Deletion analysis was therefore performed. It was found that amino acids 1-18 and 105-272 are required for interaction with these factors. The binding of BRCA2 and BRCA2a with cyclin dependent kinases was also tested using a GST-pulldown assay. BRCA2 and BRCA2a associated with cdc2 and cdk5.

[0035] Purified recombinant proteins of BRCA2 and BRCA2a were also assayed for histone acetyl transferase activity. This was done because recent studies of several enzymes involved in acetylation and deacetylation of histone residues have revealed a potential relationship between gene transcriptional activation and histone acetylation (Brownell, J. E. et al. 1996. Cell 84:843-851; Parthun, M. R. et al. 1996. Cell 87:85-94; Yang, X. J. et al. 1996. Nature 382:319-324; Orgyzko, V. V. et al. 1996. Cell 87:953-959; Mizzen, C. A. et al. 1996. Cell 87:1261-1270; Roth, S. Y. and C. D. Allis. 1996. Cell 87:5-8; Wade, P. A. and A. P. Wolfe. 1997. Curr. Biol. 7:R82-R84; Pazin, M. J. and J. Kadonaga. 1997. Cell. 89:325-328; Wolffe, A. P. 1997. Nature 387:16-17). The transcriptional activators operate by disrupting the nucleosomal structure through acetylation of histones, leading to activation of gene expression. In the present experiments, the amino terminal domain of both BRCA2 and BRCA2a demonstrated histone acetyl transferase activity. Control samples, where BRCA2 or BRCA2a was replaced with bovine serum albumin (BSA), showed no significant histone acetyltransferase activity. Similar control experiments where histones were replaced by BSA (lysine rich nonhistone protein) also failed to show significant acetyltransferase activity. These data show for the first time that BRCA2 proteins exhibit specific acetyl transferase activity to histones. Therefore, BRCA2 can be termed a histone acetyl transferase.

[0036] Since the amino terminal region of BRCA2 and BRCA2a show the histone acetyltransferase activity, it is likely that exon 3 (amino acids 18-105) which is responsible for the transactivation function of BRCA2 is not needed for histone acetyltransferase function. These data indicate that the transactivation and histone acetyltransferase functional domains of BRCA2 do not overlap with each other. In order to determine which histones are acetylated by BRCA2 proteins, the histone acetyltransferase assay was performed using free core histones and then analyzed the resulting products by SDS-polyacrylamide gel electrophoresis followed by fluorography. The results show that BRCA2 proteins acetylated primarily H3 and H4 of free histones. These results were confirmed using free histones.

[0037] The in vivo histone acetyltransferase activity of BRCA2 was then examined. Immunoprecipitation of BRCA2 from whole cell extracts was tested for acteyltransferase activity. Results demonstrated that immunoprecipitated BRCA2 carries acetylase activity specific for histones. Considered together with the in vitro data, the results support the finding that BRCA2 has intrinsic histone acetyltransferase activity, and may provide the mechanism for activation of gene expression. Patients with mutations in histone acetyltransferase or transactivation domains of BRCA2 would show a loss of gene expression which is critical for growth inhibition and differentiation, resulting in a subset of familial breast and ovarian cancers.

[0038] As with BRCA1a and BRCA1b, the shorter length of the BRCA2a splice variant makes it useful in establishing permanent cell lines for expression of this tumor suppression gene. Such cell lines will be useful model systems for studying BRCA2 gene therapy in breast, ovarian and prostate cancer cells.

[0039] Further, as with BRCA1b, the presence of this dominant negative variant, BRCA2a, may result in the dysfunction of the tumor suppressor protein BRCA2. Thus, these proteins may serve as a marker for identifying individuals with greater potential for developing breast, ovarian and prostate cancer. Altered levels of BRCA1a may also serve as a prognostic and/or diagnostic tool for breast and ovarian cancer.

[0040] Levels of the splice variants BRCA1a, BRCA1b and BRCA2a, can be routinely determined by one of skill in the art in accordance with well known methods. For example, tissue or blood samples can be screened for the presence of these splice variants by PCR, RT-PCR, Western blotting or protein truncation assays.

[0041] Several critical growth regulators like the product of the pRB, a tumor suppressor protein have been shown to associate with E2F-both in vitro and in vivo resulting in net inhibition of E2F-mediated transactivation and E2F release from pRB is an important event in the activation of genes required for S-phase entry (Weinberg, R. A. (1995) Cell, 81, 323-330). Accordingly, in vitro associations of BRCA1 and BRCA2 and splice variants thereof with E2F demonstrated herein are expected to correlate in similar fashion with in vivo activity.

[0042] The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1 Cell Lines

[0043] NIH3T3 cells and their derivatives, MCF-7, MDA-MB-453 and A431 cells were grown at 37° C. in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS); HS578 Bst cells were grown in DMEM supplemented with 10% FBS, 1% PS and 30 ηg/ml EGF, CAL-51 cells were grown in DMEM containing 10% FBS, 1% PS, 0.6 μg/ml bovine insulin, 5×10⁻³ μg/ml transferrin and 146 mg/Liter glutamine; ZR 75-1 and COLO 320 were grown in RPMI 1640 supplemented with 10% FBS, 1% PS and 10 μg/ml bovine insulin; NIH:OVCAR-3 cells were cultured in RPMI 1640 supplemented with 20% FBS, 1% PS and 10 μg/ml bovine insulin; PC12 cells were grown in RPMI 1640 supplemented with 10% horse serum and 5% FBS, 1% PS; SK-OV-3 and HBL 100 cells were grown in McCoy's 5a medium supplemented with 10% FBS and 1% PS; Saos-2 cells were grown in McCoy's 5a medium supplemented with 15% FBS and 1% PS; CAMA-1 cells were cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 10% FBS and 1% PS. All the cell lines except CAL-51 cells were obtained from The American Type Culture Collection (Rockville, Md.). CAL-51 cells were originally obtained from a patient with invasive adenocarcinoma with extensive intraductal involvement as described by Gioanni et al. (1990) Br. J. Cancer, 62, 8-13.

Example 2 Immunohistochemistry

[0044] The different cell lines in the logarithmic stage of growth cultured in chamber slides were made quiescent in the presence of DMEM only for a period of 24-72 hours. Cells were then washed in PBS and subjected to immunohistochemistry analysis using rabbit anti BRCA1 carboxyterminal or an amino terminal peptide antibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), rabbit polyclonal antibody generated against GST fusion proteins containing amino acids 1 to 76 and amino acids 1 to 331 of the BRCA1 protein in accordance with procedures described by Rao et al. (1996) Oncogene, 12, 523-528.

Example 3 Plasmid Construction and Transfections

[0045] BRCA1a cDNA and BRCA1b cDNA were subcloned into pFLAG-CMV vector (Eastman Kodak Company, Rochester, N.Y.) by PCR. Purified DNA, 5 μg for chamber slides and 20 μg for 100 mm petri dishes of pFLAG-CMV-2 expression vector or pFLAG-CMV-2 expression vector containing the BRCA1a cDNA, were transfected into COS and CAL-51 cells using the Invitrogen kit according to the manufacturer's recommendations (Invitrogen Corp., San Diego, Calif.). After 48-72 hours post transfection cells were processed for immunofluorescence analysis or the cell extracts were subjected to Western blot analysis using FLAG M2 antibody (Eastman Kodak Company, Rochester, N.Y.) in accordance with procedures described by Rao et al. (1996) Oncogene, 12, 523-528.

Example 4 Immunofluorescence

[0046] Immunofluorescence analysis was performed using pFLAG-CMV-2 BRCA1a transfected COS and CAL-51 cells cultured in chamber slides and fixed in 3.7% formaldehyde at room temperature. Slides were washed in PBS and PBS with 0.05% Triton X-100 and blocked in blocking solution (4% normal goat serum, 0.05% Triton x-100 in PBS) for 10 minutes at room temperature. The cells were incubated with primary anti FLAG M2 antibody diluted 1:100 for COS and CAL-51 cells, then washed three times with PBS, blocked with blocking solution for an additional 10 minutes at room temperature followed by incubation with the secondary antibody (FITC conjugated goat anti mouse IgG). After rinsing in PBS, the slides were mounted with mounting media and photographed on a confocal microscope.

Example 5 Preparation of Total Nuclear Extract and Western Blot Analysis

[0047] COS cells were harvested 48 hours post transfection by washing in PBS and treating with trypsin. For preparing total cell extracts, the cells were lysed in RIPA buffer and the lysate was centrifuged at 14,000 rpm for 30 minutes at 40° C. The supernatant was assayed for protein concentration by Bradford's method (Bio-Rad) and ≈50-100 μg of protein was subjected to Western blot analysis in accordance with procedures described by Rao et al. (1996) Oncogene, 12, 523-528. The nuclear extract was prepared in accordance with procedures described by Hurst et al. (1990) Molecular and Cellular Biology, 10, 6192-6203. In accordance with these procedures, cells were lysed in nuclear extract buffer 1 and centrifuged at high speed for 1 minute at room temperature. The crude nucleus was suspended in nuclear extract buffer II. Nuclear debris was removed by centrifugation for 1 minute at room temperature. The supernatant was diluted by the addition of 20 mM Hepes (pH 7.4). The protein concentrations were determined by Bradford's method (Bio-Rad) and ≈50-100 μg of protein was subjected to Western blot analysis.

[0048] For Western blotting analysis ≈50-100 μg of cell/nuclear extract in SDS sample buffer were loaded on a 10% SDS-PAGE in Bio-Rad mini-protean II cell as described previously by Rao et al. (1996) Oncogene, 12, 523-528. After electro transfer onto PVDF membrane, the FLAG-BRCA1a fusion protein was detected with anti-FLAG M2 antibody diluted 1:100 using Western exposure chemiluminescent detection system from Clonetech or ECL.

Example 6 Metabolic Labeling of Cells

[0049] Confluent 100 mm plates of HL 60 -cells were labeled with ³²P-orthophosphoric for 4 hours. The cells were lysed in radioimmunoprecipitation assay buffer. Following sedimentation the supernatants were subjected to immunoprecipitation using rabbit anti BRCA1 peptide or recombinant protein antibody or preimmune serum in accordance with procedures described by Rao et al. (1996) Oncogene, 12, 523-528. The samples were'subjected to 10% SDS polyacrylamide gel electrophoresis and autoradiography. In some cases the cold HL60 cell lysates were subjected to immunoprecipitation using carboxyterminal BRCA1 peptide antibody and then subjected to Western blot analysis using phosphotyrosine antibody (Santa Cruz Biotechnology).

Example 7 Expression and Purification of GST Fusion Protein

[0050] Log phase cultures of E. coli BL21 (DE3) LysS transformed with the pGEX 2TK-BRCA1 (aa 1-76), pGEX2T-BRCA1 (aa 1-76), pGex 2T-E2F-1, pGex2T-CycA, pGEX2T-cycB1, pGEX2T-cycD1, pGEX2T-E2F-4 plasmids were incubated with IPTG for 3 hours. The cells were pelleted in STE buffer containing 100 μg/ml lysozyme, 5 mM DTT, 1 mM PMSF and 2% Sarkosyl, sonicated on ice and centrifuged at 10,000 g for 10 minutes. Triton X-100 was added to the supernatant and applied to a glutathione sepharose 4B column (Pharmacia Biotech, Uppsala, Sweden) and the GST-BRCA1 or GST-cyclins or GST-E2F fusion proteins were either left immobilized or eluted with elution buffer containing glutathione. The GST-BRCA1 fusion proteins were labeled with ³²P in a 100 μl final volume containing 20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl₂, 10 μCi of (-³²P) ATP, 1 μg GST-fusion protein and 100 units of cAMP dependent protein kinase (Sigma Chemical Co., St. Louis, Mo.) on ice for 30 minutes.

Example 8 GST Pull Down Assay

[0051] CAL-51, ZR 75-1 or HL 60 cells were labeled with ³⁵S-methionine in accordance with procedures described by Rao et al. (1996) Oncogene, 12, 523-528. The cells were washed in cold phosphate buffered saline (PBS) and scrapped into 1 ml of TNN buffer (Takashima et al. (1994) Oncogene, 9, 2135-2144) and lysed by rotating for 30 minutes at 4° C. The lysates were centrifuged at 14,000 g for 30 minutes and subjected to protein binding assay as described by Takashima et al. (1994) Oncogene, 9, 2135-2144. For protein binding assay cell extracts were precleared overnight with GSH-beads and then incubated with either GST protein-conjugated GSH-beads or GST-BRCA1 (containing residue 1 to 76 of the BRCA1a protein). Proteins were incubated with GSH beads for 2 hours at 4° C. The beads were then washed in TNN buffer and boiled in SDS sample buffer and loaded on a 10% SDS PAGE. The gels were fixed, treated with enhance, dried and exposed to X-ray films. For in vitro binding experiments 10-20 μl of full length in vitro translated BRCA1a, BRCA1b, BRCA1 (amino acids 1-182), cdc2, cdk2, cdk4, cdk5 and E2F-1 were tested for binding to GST-E2F-1 or GST-E2F-4.

Example 9 Immunoprecipitation and In Vitro Kinase Assays

[0052] CAL-51 cells were lysed in 1 ml TNN buffer and immunoprecipitated with recombinant BRCA1 polyclonal antibody (amino acid 1-76) as described previously by Rao et al. (1996) Oncogene, 12, 523-528. The immunoprecipitates were washed in kinase buffer and measured for kinase activity toward histone H1 in accordance with procedures described by Makela et al. (1994) Nature, 371, 254-257.

Example 10 Far Western Blot Analysis

[0053] Far Western blot analysis was performed. After transfer the nitrocellulose membrane was washed in 1× HBB buffer (Singh et al. (1989) Biotechniques, 7, 252-261) and treated sequentially with 1× HBB buffer containing different concentrations of guanidine HC1 ranging in concentration from 6 M to 0.19 M. The membrane was hybridized in Hyb 75 buffer (Kaelin et al., 1992) containing 0.1 mM ZnCl₂ and ³²P-labeled GST-TK-BRCA1 protein (10⁶ cpm/ml) overnight at 4° C. Subsequently, the membrane was washed in Hyb 75 buffer, air dried and exposed to X-ray film.

Example 11 Histone Acetyl Transferase Activity Assay

[0054] The histone acetyltransferase activity (HAT) assay was carried out as described by Bannister and Kouzarides (1996. Nature 384:641-643) with slight modifications. Briefly, Crude core histones (15 μg) and GST-fusion proteins of BRCA2 and BRCA2a (50-100 ng) were mixed to give a final volume of 30 μl in buffer IPH (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% by volume NP-40, 0.1 mM PMSF). Reactions were initiated by the addition of [³H]-acetyl CoA and the reactions incubated at 3° C. for 10 to 45 minutes. Incorporation of the radiolabeled acetyl groups into histones was determined by liquid scintillation counting of reactions spotted onto P-81 Whatman filters. 

What is claimed is:
 1. A nucleic acid sequence encoding BRCA1a, BRCA1b or BRCA2a.
 2. A protein encoded by a nucleic acid sequence of claim
 1. 3. A vector comprising a nucleic acid sequence of claim
 1. 4. A host cell transfected with a vector of claim
 3. 5. A method of identifying agents capable of modulating the amount or selected activity of protein encoded by a tumor suppression gene comprising contacting cells of claim 4 with an agent suspected of modulating protein levels or activity and measuring the level or a selected activity of the protein in the cells.
 6. A method of inhibiting proliferation of breast or ovarian cancer cells in a patient suffering from breast or ovarian cancer comprising administering to the patient a vector of claim
 3. 7. A method of inducing apoptosis of breast or ovarian cancer cells in a patient suffering from breast or ovarian cancer comprising administering to the patient a vector of claim
 3. 8. A method of suppressing tumors in a patient suffering from breast or ovarian cancer comprising administering to the patient a vector of claim
 3. 9. A method of identifying individuals at greater risk for developing breast or ovarian cancer comprising detecting a protein of claim 2 in an individual.
 10. A method of increasing histone acetyltransferase activity in breast and ovarian cancer patients with decreased levels of histone acetyltransferase activity comprising administering to the patient a vector containing the nucleic acid sequence of BRCA2 so that histone acetyltransferase activity is increased.
 11. A method of identifying individuals at greater risk for developing breast, ovarian or prostate cancer comprising detecting decreased levels of BRCA2 protein in an individual. 